Clone、construction and expression of Synechocystis sp.PCC 6803 Acetyl- CoA carboxylase gene for fat production

在化石燃料日益枯竭、环境污染逐渐威胁人类生存的今天，生物柴油在提供可再生能源的同时还可以减少温室气体，从而受到世界各国的广泛重视。蓝藻为光合自养微生物，是大气碳循环主要贡献者，其生物柴油产量是油料作物的8～24倍。乙酰辅酶A羧化酶(acetyl-coenzymeAcarboxylase，ACCase)是生物体内脂肪生物合成的关键酶，研究发现，过量表达ACCase能够促进生物体脂肪的合成。相对于蓝藻，大肠杆菌具有遗传背景清楚、生长速度快等特点，利用基因工程构建的大肠杆菌表达系统是目前最常用的外源基因表达系统，在转基因生产中占据着主导地位。 本文以集胞藻Synechocystissp.PCC68...With the increasingly severe problems of energy crisis and environmental pollution, biodiesel has been attracting more and more attention worldwide by providing renewable energy and reducing greenhouse emissions. As photoautotrophic microorganisms, Cyanobacteria are major contributors to atmospheric carbon cycle, the biodiesel productivity of which is 8 to 24 times of that observed in oilseeds. Ac...学位：理学博士院系专业：生命科学学院_微生物学学号：2162006015330

超广谱内酰胺酶研究进展

超广谱β-内酰胺酶(extended specturn β-lactamerses,ESBLs)是丝氨酸蛋白酶的衍生物,它能够水解青霉素、广谱及超广谱头孢菌素和单环β-内酰胺抗生素的β-内酰胺酶,且能被克拉维酸抑制。ESBLs主要由肠杆菌科细菌产生,

三种检测方法对样品中沙门氏菌的检测结果比较

本实验采用VIDAS、API20E和SN0170-92三种不同的检测方法对CNCA举行的食品中常见致病菌能力验证计划中提供的一份盲样进行检验,总结对比了3种不同检验方法对沙门氏菌检测的各自优缺点

Loop-Mediated Isothermal Amplification(LAMP) for Detection of Alicyclobacillus acidoterrestris in Foods

目的:利用环介导等温扩增技术建立食品中酸土环脂芽孢杆菌快速检测方法。方法:针对酸土环脂芽孢杆菌16S序列设计特异引物,再优选反应体系,用显色法检测实验结果。结果:该方法能够在63℃条件下1 H内检出食品中酸土环脂芽孢杆菌,所设计的引物有良好的特异性;灵敏度达6.7 Cfu/M l(弱阳性)。结论:该方法具有高效、特异性强和敏感性高等特点,可满足酸土环脂芽孢杆菌快速检测筛选的要求。Purpose: A loop-mediated isothermal amplification(LAMP) method was established for the detection of Alicyclobacillus acidoterrestris in foods.Methods: After optimization of the reaction conditions of LAMP including the concentrations of primers, reaction time and amplification temperature, the LAMP method was developed, and its sensitivity and specificity were evaluated.Results: The method was capable of rapidly and specifically detecting A.acidoterrestris in foods within 1 hour at a constant temperature of 63 ℃.The sensitivity of the method was 6.7 CFU/m L and the specificity was 100%.Conclusions: The LAMP method is efficient, highly sensitive and specific, and suitable for the rapid detection of A.acidoterrestris in various food samples.福建省漳州市自然科学基金项目(ZZ2012J16

Human Cu，Zn－SOD Gene Mutagenesis and Expression in Algae and Activity Study

超氧化物歧化酶（Superoxidedismutase,SOD）的主要功能是催化歧化超氧阴离子自由基(O2－.),由于SOD能清除生物体内的O2－.˙,所以在防御氧的毒性、抗辐射损伤、预防衰老以及防治炎症和肿瘤等方面都有重要作用。但SOD在体内的稳定性比较差、半衰期比较短，从而使其功能的发挥受到一定的限制。本研究对pSOD质粒中的人铜，锌SOD（hCu,Zn-SOD）基因进行定点突变,将Cys111密码子突变为Ala111密码子，再分别与热休克groESL启动子、rbcSpolyA终止区和Kanr基因连接，构建为pESODT111质粒。然后再连接上蓝藻Synechococcussp.PCC79...The main function of superoxide dismutase(SOD) is to eliminate the superoxide free ion(O2－.).So it is widely applied to protect human body from the hurt of oxygen,radiation and postpone consenescence etc. However, the application of Cu,Zn-SOD is limited for its short half life in vivo. The Cys111 genetic code of hCu,Zn-SOD gene in the pESOD plamid was mutated into the Ala111 code with sited-di...学位：理学硕士院系专业：生命科学学院生物学系_生物化学与分子生物学学号：20012603

Advances of research of Cyanobacterium genome

本文对近年来蓝藻基因组研究进展进行了综述.介绍了聚胞藻PCC6803基因组研究方法和成果,包括最佳分析软件的选择,所获得的基因组基本信息,以及后基因组研究的部分成果.蓝藻基因组间差异很大,文中介绍了其它蓝藻基因组的基本信息和在各个藻种里的重要发现.文章最后探讨了蓝藻基因组的研究意义和前景.The recent research advances of Cyanobacteria genome were reviewed in this paper. As the representative of genome of Cyanobacteria, the research　methods and results of genome of Synechocystis sp.strain PCC6803 wereintroduced, including the selection about most suitable analyzing soft, the genome message and some results of its post-genome research. For the much difference among cyanobacteria, the basic message and significant discovery of others Cyanobacteriagenome were described. Furthermore, the research meaning and future develoment of the Cyanobacteria genome were discussed

Cloning,sited-directed mutagenesis and expresison of hCu,Zn-SOD gene in E.coli

目的　在克隆人源铜锌超氧化物歧化酶基因 (hCu ,Zn -SOD)的基础上 ,对hCu ,Zn -SOD进行定点突变 ,使其在E .coliDH5α中表达 ,为进一步改造hCu ,Zn -SOD基因奠定基础。方法　首先构建质粒 pESOD ,然后用定点突变技术把其中hCu ,Zn -SOD的Cys111密码子突变为Ala111密码子 ,再与 pUCMT1相连接 ,构建表达载体pE SODT111,使其在E .coliDH5α中表达 ,表达产物用改良的邻苯三酚法和Westem杂交测定。结果　hCu ,Zn -SOD突变后在E .coliDH5α中成功表达 ,表达产物具有SOD活性 ,活力为 16 4 4 7U/ml培养液。结论　hCu ,Zn -SOD基因经过定点突变后构建的表达载体可在DH5n中表达 ,表达产物同样具有天然的hCu ,Zn -SOD活性Objective For further researching of hCu,Zn-SOD gene engineering,to mutate human copper,zinc-superoxide dismutage gene(hCu,Zn-SOD),and expressed the plamid in E.coli DH5α.Methods The pESOD plamid was constructed firstly,then the Cys 111 genetic code of hCu,Zn-SOD gene in the pESOD plamid was mutated into the Ala 111 code with sited directed mutagenesis and the expression plamid pESODT111 was constructed by inserting the mutated pESOD into pUCMT1 plamid.The pESODT111 plamid was expressed in E.coli JM101.The expression product was determined by Western blot and improved by pyrogallol autoxidation. Results Mutated hCu,Zn-SOD gene expressed in E.coli DH5α correctly,the expression product had SOD activity-16.447?U/ml culture medium.Conclusion The expression product of the mutated hCu,Zn-SOD had activity of native hCu,Zn-SOD.国家自然科学基金 (40 2 0 60 1 8

Sited-directed mutagenesis of hCu,Zn-SOD gene and its expression in Synechococcus sp.PCC7942

应用PCR定点突变技术把质粒pESOD中人铜锌超氧化物歧化酶基因(hCu,Zn-SOD)的Cys111密码子突变为Ala111密码子,再构建重组子,通过随机同源重组将突变后的hCu,Zn-SOD整合入聚球藻Synechococcussp.PCC7942,并实现表达。表达产物用SDS-PAGE、Western blot、酶活等方法测定均为阳性反应;热稳定性测定显示,hCu,Zn-SOD在80℃保温30min后仍具有95%的活力,耐热能力比天然hCu,Zn-SOD有了较大的提高。蛋白扫描结果显示目的蛋白占可溶性蛋白的3.61%。The Cys~(111) genetic code of human copper/zinc superoxide dismutase(hCu,Zn_SOD) gene in the pESOD plamid was mutated into the Ala~(111) code with site_directed mutagenesis,and then the plamid pESODT~(111) which contained groESL promoter,mutated hCu,Zn_SOD gene,rbcS_polyA terminator and reporter gene(Kan~r) was constructed and transduced into Synechococcus sp.PCC7942 with homologous recombination platform.The results of PCR and DNA sequence analysis showed that the target nucleotide had been genetically integrated into genome DNA of the host cell.SDS_PAGE,Western blot and Pyrogallol autoxidation assay confirmed that the transformant strains expressed the mutated hCu,Zn_SOD protein.And the level of the mutated hCu,Zn_SOD protein reached a value of 3.61% of the total soluble protein.Furthermore,the transformants still retained 95% activities of SOD after 30 minutes at 80℃ environment,it indicated that the mutated hCu,Zn_SOD protein could endure higher temperature than the natural one.福建省自然科学基金(C0510004)~